Fibrin(ogen) derivatives, process for their preparation and their use

ABSTRACT

Oxidized derivatives of-fibrin and fibrin or fibrinogen degradation products or partial sequences, process for their preparation and their use as medicaments, for diagnosis or as an affinity agent are described.

This is a continuation of application Ser. No. 07/787,780, filed Jul.18, 1991, now U.S. Pat. No. 5,376,631, which is a continuation ofapplication Ser. No. 07/364,351, filed Jun. 9, 1989, abandoned.

The invention relates to a process for the preparation of derivatives offibrin, or fibrin or fibrinogen degradation products or partialsequences, wherein these are treated with an oxidizing agent, productsprepared by this process and their use as medicaments or for diagnosis.

Human blood possesses an enzymatic system which is capable ofredissolving blood clots which have formed: the fibrinolytic system.

The central enzyme in fibrinolysis, plasmin, is formed from itsprecursor plasminogen via activators which are liberated fromendothelial cells and other cell associations. The activators can besubdivided into two types: on the one hand the plasminogen activators ofthe tissue type (termed t-PA) and on the other hand the plasminogenactivators of the urokinase type (u-PA).

T-PA has a high affinity for fibrin, heparin, and plasmin or BrCN-degradation products of fibrinogen, and its plasminogenolytic activityis stimulated in the presence thereof.

T-PA stimulators of this type are of great interest in the therapeuticand diagnostic respect. They enable considerable dosage reductions int-PA robe administered and promote a sensitive t-PA detection in plasma.In addition, they allow simple affinity chromatographic purifications oft-PA from biological fluids, for example cell supernatant.

Stimulators such as BrCN-degradation products of fibrinogen can,however, not be employed for therapeutic use because of their hightoxicity. In vivo heparin has a low efficiency as a stimulator for t-PA.

Polymorphonuclear leukocytes and macrophages participate inphysiological fibrinolysis by secretion of proteinases, inter aliaurokinase, collagenase and elastase, and also of oxidation products, inthis case in particular N-chloramines.

Surprisingly it has now been found that the oxidation of fibrin andfibrin or fibrinogen degradation products (FDP) caused by N-chloramines,such as chloramine T, leads to a substantial (about 5-fold to 10-fold)improvement in the t-PA-stimulating properties of these products(stimulation of unoxidized FDP=approximately 5-fold and stimulation ofoxidized FDP=approximately 25-fold, compared with t-PA withoutstimulator). It was found that the D region of fibrinogen is the domainresponsible for this effect.

The invention therefore relates to derivatives of fibrin, or fibrin orfibrinogen degradation products or partial sequences, wherein all orsome of the methionine residues in the basic amino acid sequences havebeen converted to methionine sulfoxide residues.

The invention also relates to a process for the preparation ofderivatives of fibrin, or fibrin or fibrinogen degradation products orpartial sequences, which comprises treating fibrin, or fibrin orfibrinogen degradation products or partial sequences, with an oxidizingagent.

The invention also relates to a process wherein fibrinogen or fibrin istreated with an oxidizing agent and the product is degraded. Enzymaticdegradation is preferred, specifically, for example, by the action ofplasmin, thrombin or batroxobin on oxidized fibrinogen.

Soluble fibrin (so-called fibrin monomers) as starting material isprepared, for example, by incubating 1 mg/ml factor XIII-freefibrinogen, for example F XIII reagent, Behringwerke, in 25 mmol/l Tris,5 mmol/l CaCl₂ and 2 mmol/l Gly-Pro-Arg-Pro, pH 7.8, for 10 minutes at37° C. with 0.5 IU test thrombin and adding 5 IU hirudin/ml.

Fibrinogen degradation products can be produced in accordance with theprocess described by Stief et al. (Thromb. Res. 48, 603-609, 1987). Afibrinogen solution (1-10 mg/ml) in 25 mmol/l Tris and 5 mmol/l EDTA, pH7.8, is degraded by the action of plasmin , preferably by passing thefibrinogen solution through a plasmin sepharose (2.5 mg protein/ml gel)at a flow rate of about 60 ml/h.

The oxidation of the starting materials (=fibrinogen solution or thefibrin monomers or degradation products formed) is carried out at pH7-9.5, preferably in a buffer, in particular Tris buffer, if appropriatewith the addition of 0.01-0.2 and preferably 0.1% (w/v) detergent, suchas ^(R) Triton×100, and, if appropriate, with the addition of mannitol,preferably 5-20mmol/l, to improve the solubility. The oxidizing agentused is preferably a chloramine, such as chloramine T, specifically in aconcentration of 1-20 mmol/l, preferably 5-10 mmol/l. The oxidation canbe terminated by adding an excess of reducing agent, such as ascorbicacid, acetylcysteine or acetylmethionine. The oxidation can be carriedout at 4-45° C., preferably 20°-40° C., and proceeds within seconds(less than 5 min).

The end products are characterized by amino acid analysis. After proteinoxidation, methionine is detected only in traces whereas methioninesulfoxide is detected in amounts which correspond to the methioninecontent occurring in the protein. The plasmin-degraded and oxidizedderivatives also possess a better solubility in distilled water than theunoxidized derivatives.

Synthetic methionine-containing peptides containing sequences from theD-region of fibrinogen are also suitable as starting material for theprocess according to the invention. Oxidized FDP bind t-PA and sc-uPA(single chain form urokinase) with a higher affinity than unoxidizedFDP, as shown in tests with FDP covalently bonded to ^(R) Sepharose 4B.For this reason oxidized fibrin and fibrinogen derivatives areparticularly suitable for purifying t-PA or sc-uPA (for example fromcell supernatants) by affinity chromatography.

Suitable oxidizing agents are, in particular, methionine-specificoxidants, which preferably convert methionine to methionine sulfoxide inan alkali medium, preferably chloramines (for example chloramine T orchloramine B), or salts of hypochlorous acid (for example NaOCl).According to Savige and Fontana (Methods in Enzymology 47: 453-459,1977), dye (for example methylene blue)-mediated photooxidation and alsobromosuccinimide; N-chlorosuccinimide;2,4,5-tribromo-4-methylcyclohexadienone; BNPS-skatole(2-(2-nitrophenylsulfonyl)-3-methyl-3- bromoindolamine); chloramine T;t-butyl hypochlorite; trichloromethanesulfonyl chloride and also1-chlorobenzotriazole, iodobenzene dichloride in aqueous pyridine,pyridine bromide complexes, quinolines and, in particular,1,4-diazobicyclo(2.2.2)octanes in aqueous acetic acid are suitable forthe conversion of methionine to methionine sulfoxide.

The oxidation, preferably at pH 8-9, is improved by the addition of0.05-0.1% detergent, such as ^(R) Triton×100.

Oxidized FDP likewise bind to fibrin with a considerably higher affinitythan non-oxidized FDP, as can be shown in tests with clotted plasma. Forthis reason they are particularly suitable for directing substancesvaluable for thrombus diagnosis and therapy to a clot in vivo.

According to the state of the art, thrombi are detected in vivo usingradioactively labeled fibrinogen (Mahn, I., and Muller-Berghaus, G.,Haemostatis, 4: 40:50, 1975). Since oxidized FDP possess a considerablyhigher affinity for the thrombus than fibrinogen, the use ofradioactively labeled oxidized FDP, preferably FDP-D, is moreadvantageous since 1. the clinical findings are available within ashorter time (less than 1 h) after administration of the diagnosticagent and 2. the total organism is subjected to lower radioactive dosessince less labeled protein is required because of the high affinity.

On the other hand, according to the state of the art, plasminogenactivators (PA) are transported to fibrin clots by using PA covalentlybonded to fibrin antibodies or chimeras of PA and fibrin antibodiesproduced by gene technology.

Disadvantages of these methods lie in the induction of antibodiesagainst the protein foreign to the body. The side effects associatedherewith (for example immune complex diseases) can be avoided by the useof human oxidized FDP. HOCl--and chloramine-producing enzymes, such asmyeloperoxidase, glucose oxidase and xanthine oxidase can also betransported to a thrombus by means of oxidized FDP if they are in acovalently bonded state with oxidized FDP. The enzyme reaction can bestarted by infusion of suitable non-toxic substrates (for exampleglucose, xanthine, hypoxanthine). Oxidation of fibrin polymers in vivolowers the requirement for endogenous and, if necessary, foreign t-PA todissolve a thrombus, as a result of which, only small amounts of t-PAhave to be administered for a successful lysis. The associated oxidativeinactivation of the fibrinolysis inhibitor alpha-2-antiplasmin bonded inthe thrombus intensifies the profibrinolytic effects.

In addition, intravenous administration of oxidized FDP gives rise to anintensified generalized stimulation of t-PA and sc-uPA, as a result ofwhich the dose of this PA can be reduced or its use can be dispensedwith entirely.

Furthermore, oxidized fibrin(ogen) derivatives are suitable for asensitive and specific detection of plasminogen activators which can bestimulated, such as t-PA, in in vitro test systems in plasma or otherbiological fluids. For this purpose, for example, 50-200 μg of oxidizedfibrinogen degradation products/ml reaction batch are used in achromogenic detection method for t-PA together with 3 CTA-U plasminogenand 0.3 μM of a chromogenic plasmin substrate.

EXAMPLE 1

Preparation of the oxidized fibrinogen degradation products

a) Plasmin degradation products 250 mg of test fibrinogen (Behringwerke)dissolved in 50 ml of distilled water with 5 mmol/l EDTA were passedthrough 25 ml plasmin- ^(R) Sepharose 4B (2.5 mg plasmin/ml gel) at aflow rate of 60 ml/h at room temperature (RT). The effluent (Eff.) wasadjusted to pH 8.5, 12.5 ml of 100 mol/l chloramine T were added and themixture was incubated for 15 minutes at 37° C. The oxidation batch wasthen dialyzed (48 h, 4° C.) against the 200--fold volume of 50 mmol/lTris, pH 8.5.

Alternatively, the oxidized degradation products could be obtained by anoxidation taking place prior to passage through the plasmin-sepharoseand subsequent plasmin proteolysis.

b) Thrombin degradation products Thrombin degradation products,according to the invention, of oxidized fibrinogen are obtained by firstoxidizing 1 mg/ml fibrinogen with chloramine T at a concentration of 2.5mmol/l and then incubating with 0.5 IU test thrombin (Behringwerke) for10 minutes at 37° C. and pH 7.8 and adding 5IU hirudin/ml.

EXAMPLE 2

Stimulation of the plasminogenolytic activity of t-PA by FDP as afunction of the oxidizing agent concentration--detection of t-PA byoxidized FDP

a) 100 μg of FDP-BrCN (FDP prepared via BrCN degradation) and,respectively, 100 μg of FDP-EDTA (FDP prepared via plasmin degradationin the presence of EDTA) in 100 μl of 50 mmol/l Tris, 100 mmol/l NaCl,0.01% ^(R) Triton ×100, pH 8.5, and Tris buffer without FDP wereincubated with 50 μl of chloramine T of various concentrations (0-20mmol/l) in distilled water for 10 min at 37° C. After adding 100 μl of3mmol/l HD-Nva-CHA-Lys-pNA in distilled water, 200 μl of 1.3 μmol/lGlu-plasminogen in 100mmol/l Tris, 100 mmol/l NaCl, 1% Haemaccel and0.1% ^(R) Triton×100, pH 8.4 (TNHT buffer), and 10 IU two-chain t-PA in200 μl of TNHT buffer, a further incubation for 12 min at 37° C. tookplace. The conversion of the substrate was terminated by adding 500 μlof 3.4 mol/l acetic acid and the resulting change in absorption at 405nm was detected.

b) In a variation, the oxidation of the FDP by addition of 50 μl of 10mmol/l chloramine T was terminated after 10 min (37° C.) pre-incubationby adding 50 μl of 5 mmol/l dithiotreitol and the plasminogen activationwas then carried out.

Result from Example 2 a, b:

                  TABLE 1                                                         ______________________________________                                        Fibrinogen degradation products                                               Chloramine T          with EDTA                                               (concentra-           through                                                 tion in    BrCN-de-   plasmin-                                                50 μl   degraded   seph.       without FDP                                 addition) Plasmin activity (A.sub.405 nm ; mE)                                ______________________________________                                        0   mmol/l     898        1053      122                                       1   mmol/l     1007        947      132                                       2.5 mmol/l     1067       1310      134                                       5   mmol/l     914        1936      135                                       7.5 mmol/l     728        2218      136                                       10  mmol/l     517        2315      132                                       15  mmol/l     220        2091      118                                       20  mmol/l     138        1900       98                                       10  mmol/l/                                                                   5   mmol/l DTT 806        2356      129                                       ______________________________________                                    

Increasing concentrations of chloramine T lead to an increased capacityfor t-PA stimulation. FDP-EDTA and FDP-BrCN show an improvement in theirstimulation of t-PA which is only minimal (10%) and is only found at lowchloramine T concentration. Higher chloramine T concentrations lead toan impairment in the t-PA stimulation.

EXAMPLE 3

Stimulation of the plasminogenolytic activity of t-PA by differentFDP^(s) - t-PA detection by FDP

100 μg each of FDP-BrCN, FDP-EDTA, fibrinogen and FDP-EDTA obtained bypassing oxidized fibrinogen (see Example 1) through a plasmin-^(R)Sepharose were examined in accordance with the test conditions listed inExample 2a) with the addition of 50 μl of 20 mmol/l chloramine T anddistilled water to check their stimulating power towards two-chain t-PA.The oxidation was terminated after 10 min (37° C.) by adding 100 μl of6.25 mmol/l dithiotreitol and the substrate conversion was terminatedafter 9 min. (37° C.) by adding 500 μl of 3.4 mol/l acetic acid.

See Table 2 for the result.

In the case of FDP-EDTA, oxidation in the test leads to a pronouncedrise in the t-PA-stimulation power. Before oxidation in the assay,FDP-BrCN and FDP-EDTA from preoxidized fibrinogen already possess morethan 10 times the stimulation power as compared with the buffer control.The oxidation of fibrinogen also increases its stimulation potential.

                  TABLE 2                                                         ______________________________________                                                             50 μl of 20 mmol/l                                               Distilled water                                                                         chloramine T                                             Stimulant    Plasmin activity (A.sub.405 nm ; mE)                             ______________________________________                                        FDP-BrCN     443         614                                                  FDP-EDTA     276         1172                                                 FDP-EDTA     485         962                                                  (from pre-                                                                    oxidized                                                                      fibrinogen)                                                                   Fibrinogen   117         156                                                  Buffer control                                                                              30          27                                                  ______________________________________                                    

EXAMPLE 4

Affinity chromatographic purification of t-PA and sc-uPA by immobilizedoxidized fibrin or fibrinogen degradation products and derivatives

1 ml aliquots of single chain (sc)-t-PA, sc-uPA and two-chain (tc)-uPA(1 μg/ml) in TNHT buffer were incubated for 45 min. at room temperaturewith, in each case, 0-300 μl of a 50% strength FDP-EDTA solution oroxidized FDP-EDTA-^(R) Sepharose 4B (2 mg protein/ml gel) with a gentleshaking. After centrifugation, the supernatants were removed and testedfor their plasminogenolytic activity. The residual activity wasexpressed in percent of the initial activity. 100 times the amount ofTNHT buffer was added to the ^(R) Sepharose 4B remaining in the sedimentand the whole was mixed thoroughly and centrifuged again. Thesupernatant was decanted off and 1 ml of eluent was added. 2 M KSCN and1% sodium dodecyl sulfate were used for this purpose. 10 minutesincubation (RT) with shaking were followed by centrifugation anddialysis (48 h, 4° C.) against 200 times the volume of TNHT buffer. Thesamples were then tested for their plasminogenolytic activity.

Plasminogenolytic activities were determined by incubating 50 μl ofsample (supernatant liquor) with 100 μg of FDP-BrCN, 278 pM ofGlu-plasminogen and 0.3 μmol of HD-Nva-CHA-Lys-pNA in a volume of 600 μlfor 5 min at 37° C. The conversion of the substrate was terminated byadding 500 μl of 3.4 mol/l acetic acid and the resulting extinction at405 nm was determined.

Result:

Immobilized, oxidized FDP-EDTA show a considerably higher affinity fort-PA than unoxidized FDP. In contrast to u-PA, sc-uPA shows an affinityfor bonding to oxidized FDP. The elution of the bound PA molecules canbe effected by 2 mol/l KSCN or 1% SDS.

                  TABLE 3                                                         ______________________________________                                                       Residual activity in the                                       Oxidized (non-oxidized)                                                                      supernatant liquor                                             50% strength FDP-                                                                            sc-t-PA   sc-u-PA   tc-u-PA                                    sepharose (μl) added                                                                      (%) of the initial activity                                    ______________________________________                                         0             100 (100) 100 (100) 100 (100)                                   20             97 (100) 100 (100) 100  (98)                                   50             85 (100)  94 (100) 100 (100)                                  100             55  (87)  91  (97)  97 (100)                                  200             47  (81)  78  (92)  96  (99)                                  300             31  (47)  55  (90)  90  (96)                                  ______________________________________                                    

EXAMPLE 5

Separation of the oxidized and non-oxidized fibrinogen degradationproducts through DEAE-Sephacel and subsequent polyacrylamide gelelectrophoresis (PAGE) and assay of stimulation by the individualfragments.

The effluent from the plasmin-sepharose, as indicated in Example 1, wasre-dialyzed against 200 times the volume of 10 mmol/l sodium phosphatebuffer, pH 8.6. The dialysate was then applied to 100 ml of theDEAE-Sephacel pre-equilibrated with three times the column volume of 10mmol/l sodium phosphate. Elution (10 mmol/l sodium phosphate, pH 8.6,and 300 mmol/h potassium hydrogen phosphate, pH 4.3, 100 ml in eachcase) was then carried out at a flow rate of 60 ml/h at room temperatureby means of an increasing ionic strength and decreasing pH gradient. 3ml fractions were collected.

Effluent (before applying of the gradient) and individual peaks wereexamined with respect to optical density at 280 run and also in sodiumdodecylsulfate PAGE (see Tables 4 and 5 for results). Fibrinogendegradation products by plasmin degradation possess the followingapproximate molecular weights(KDa): fibrinogen 340, X 300, Y 150, D 90,87, 85, E 60.

                  TABLE 4                                                         ______________________________________                                        Non-oxidized starting material                                                                          Main bands (subsidi-                                                          ary bands) in SDS                                   Fraction No.   OD.sub.280 nm                                                                            gel, unreduced (KDa)                                ______________________________________                                        Effluent                                                                              14-29      0.7        90                                              Peak 1  47-50      4.4        90, 85                                          Peak 2  51-54      5.6        90 (300, 60, 150)                               Peak 3  55-58      0.46       90, 60 (300, 150)                               Peak 5  83-89      0.3        60, 300 (150)                                   Peak 6  90-98      0.32       60 (300)                                        Peak 7  109-113    0.1        (90, 300)                                       ______________________________________                                    

Peak I corresponds to fibrinogen fragment D.

                  TABLE 5                                                         ______________________________________                                        Oxidized starting material                                                                              Main bands (subsidi-                                                          ary bands) in SDS                                   Fraction No.   OD.sub.280 nm                                                                            gel, unreduced (KDa)                                ______________________________________                                        Effluent                                                                              12-30      0.34       (90, 60, 150, 300)                              Peak 1  53-55      0.4        90                                              Peak 2  56-60      0.98       85                                              Peak 3  61-67      2.2        90                                              Peak 4  85-98      0.72       60, 300, 150                                    Peak 5  109-120    0.08       300 (90)                                        ______________________________________                                    

Peaks 1-3 correspond to fibrinogen fragment D.

100 μl of the individual fractions from the DEAE separation ofunoxidized FDP (EDTA) were examined in accordance With Example 2 fortheir t-PA-stimulating activity in the unoxidized and oxidized state.See Table 6 for results.

                  TABLE 6                                                         ______________________________________                                                   a) distilled                                                                              b) 20 mmol/l                                                      water       chloramine T                                           Addition of 50 μl                                                                       Plasmin activity A.sub.405 nm /9 min (mE)                        ______________________________________                                        Effluent (main                                                                             184            703                                               band in SDS gel)                                                              Peak 1 (FDP-D)                                                                             320           1890                                               Peak 2 (FDP-D)                                                                             451           1728                                               Peak 3 (FDP-D, E)                                                                          583           1145                                               Peak 5 (E, X)                                                                              266            223                                               Peak 6 (E)   197            291                                               ______________________________________                                    

It is recognized from the measured plasmin activities that the domain ofthe fibrinogen molecule which is responsible for the stimulation and forthe oxidation intensified stimulation is the D region.

EXAMPLE 6

Amino acid analysis of oxidized and unoxidized FDP-D

1 mg/ml of fibrinogen degradation product D (peak 1, purified inaccordance with Example 5) was incubated in accordance with Example 1 in50 mmol/l Tris buffer, pH 8.5, with and without the addition of 5 mmol/lchloramine T for 30 min at 37° C. This was followed by dialysis against100 times the volume of distilled water for 48 h at 4° C. and the use of1.2 nM protein per analysis in a LKB 4150 amino acid analyzer undervaried standard hydrolysis conditions (24 h, 110° C. in vacuo, 3 Np-toluenesulfonic acid). The results are mean values of tripledeterminations. The amino acid tryptophan could not be determined underthe conditions used (Table 7).

                  TABLE 7                                                         ______________________________________                                        Amino acid composition                                                                           D (%)   oxid. D (%)                                        ______________________________________                                        Asp                13.2    12.5                                               Thr                4.0     4.3                                                Ser                5.8     6.1                                                Glu                10.3    12.4                                               Pro                4.9     5.6                                                Gly                11.6    12.2                                               Ala                4.7     4.5                                                Cys                0.4     0.4                                                Val                3.9     4.4                                                Met                0.8     <0.1                                               Ile                3.3     3.5                                                Leu                5.3     5.6                                                Tyr                2.8     2.4                                                Phe                3.1     3.0                                                His                3.2     3.3                                                Lys                8.4     8.2                                                Arg                6.3     6.9                                                ______________________________________                                    

The two proteins differ essentially in the lack of methionine inoxidized D. Methionine sulfoxide migrates in the analyzer to anothersite and is not indicated as methionine.

EXAMPLE 7

Increased affinity for fibrin thrombi of oxidized FDP compared withunoxidized FDP: detection of thrombi

0.5 ml of plasma was clotted by adding 50 μl of 0.2 mol/l CaCl₂ and 0.5IU of thrombin. The resulting clots were then washed by centrifugingthree times in 20 times the amount of phosphate buffered saline (PBS)and incubated for one hour at room temperature in, in each case, a PBSsolution of 0.5 ml of oxizided FDP (FDP=plasmin degradation offibrinogen in the presence of 5 mmol/l EDTA) (1 mg/ml) and 0.5 ml ofunoxidized FDP (1 mg/ml) or, respectively, 0.5 ml of fibrinogen (1mg/ml) (control=FDP solutions without clots). Subsequently the residualstimulation power of the FDP in the supernatant liquor was tested byincubating 50 μl of a tissue plasminogen activator solution (500 IUtc-t-PA/ml) in PBS with 100 μl of supernatant liquor, 200 μl ofplasminogen (=1 CTA-U in 100 mmol/l Tris, 100 mmol/l NaCl 1% (w/v) ^(R)Haemaccel, 0.1% (w/v) ^(R) Triton×100, pH 8.4) and 100 μl of 3mmol/lD-norvalyl-cyclohexyl-alanyl-lysyl-paranitroanilide (HD-Nva-CHA-Lys-pNA)in distilled water for 8 min at 37° C., stopping with 500 μl of 8.5mol/l acetic acid and determining the extinction at 405 nm.

See Table 8 for result.

                  TABLE 8                                                         ______________________________________                                                      Plasmin activity                                                              (t-PA)                                                          Sample = stimulus                                                                           A.sub.405 nm /8 min                                                                       t-PA stimulation                                    (1-3)         (mU)        power*                                              ______________________________________                                        Without clots:                                                                1 oxid. FDP   1,235 + 14  15.4                                                2 unoxid. FDP 587 + 6      7.3                                                3 Fibrinogen  216 + 1      2.7                                                With clots:                                                                   1 oxid. FDP   755 + 4      61%**                                              2 unoxid. FDP 465 + 1     80%                                                 3 Fibrinogen  171 + 2     80%                                                 Control (stimulus)                                                                           80 ± 3                                                      percentage                                                                    ______________________________________                                         *Ratio of tPA activity with stimulus: tPA activity without stimulus           **in % without clots                                                     

It can be seen that the greatest decrease an the t-PA stimulation powerof the supernatant liquor occurs with oxidized FDP, which means thatoxidized FDP has a higher binding affinity to fibrin than unoxidized FDPor fibrinogen.

EXAMPLE 8

Oxidation of fibrin monomers leads to an intensification of theirstimulation properties towards t-PA

100 μl (1 mg/ml) of fibrin monomers in buffer were incubated withchloramine T of different concentrations for 10 min. at 37° C. Theoxidation was then terminated by adding 100 μl of mmol/lN-acetyl-methionine, pH 8.4, and

200 μl of tc-t-PA (=10 IU) in buffer , 200 μl of plasminogen (1 CTA-U)in buffer and 100 μl of 3 mmol/l HD-Nva-CHA-Lys-pNA in distilled waterwere added and the mixtures incubated for 24 min. at 37° C.

The substrate conversion was terminated by adding 500 μl of 8.5 mol/lacetic acid and the resulting extinction at 405 nm was determined.

                  TABLE 9                                                         ______________________________________                                        Result:                                                                       Chloramine T addition                                                                         Plasmin activity (mA)                                         (50 μl) (mmol/l)                                                                           fibrin monomers                                                                            buffer                                           ______________________________________                                         0              1274 + 5     244 + 3                                           1               551 + 5                                                       5               795 + 11                                                     10               2095 + 45                                                    20              2287 + 4                                                      40              2276 + 4     248 + 8                                          ______________________________________                                    

It can be seen that increasing concentrations of chloramine after aninitial decrease subsequently leads to an increase in the t-PAstimulation. Addition of methionine does not impair the measurementmethod, as can be shown in the buffer control.

I claim:
 1. A process for affinity chromatographic purification of aplasminogen activator from a biological fluid comprising the stepsof:incubating a solution containing a plasminogen activator with aderivative wherein the plasminogen activator is bound to the derivativeand wherein said derivative is a derivative of fibrin, fibrindegradation products or fibrinogen degradation products, or partialsequences thereof, comprising the D-region of fibrinogen, whereinmethonine residues in the basic amino acid sequence have been convertedto methionine sulfoxide using an oxidant under conditions in which theoxidant is specific for methionine, and wherein essentially no tyrosineresidues are iodized; separating the bound plasminogen activator fromthe biological fluid; and eluting said bound plasminogen activator.
 2. Aprocess for the preparation of a derivative of fibrin, fibrindegradation products or fibrinogen degradation products, or partialsequences thereof, comprising the D-region of fibrinogen, whichcomprises converting methionine residues in the basic amino acidsequence to methionine sulfoxide using an oxidant under conditions inwhich the oxidant is specific for methionine and in which essentially notyrosine residues are iodized,wherein the oxidation is carried out inthe presence of octoxynol.
 3. A process for the detection of thepresence of t-PA in a biological fluid or the determination of theamount of t-PA in a biological fluid, which comprises:i) adding aderivative of fibrin, fibrin degradation products or fibrinogendegradation products, or partial sequences thereof, comprising theD-region of fibrinogen, wherein methionine residues in the basic aminoacid sequence have been converted to methionine sulfoxide using anoxidant under conditions in which the oxidant is specific formethionine, and wherein essentially no tyrosine residues are iodized, toa reaction mixture comprising (a) a sample of said biological fluid, (b)plasminogen, and (c) a chromogenic plasmin substrate; and (ii) detectingthe presence or determining the amount of said t-PA based upon thedeveloping color.